The innate immune system displays a series of Pattern Recognition Receptors (PRRs) that detect Pathogen Associated Molecular Patterns (PAMPs) on microorganisms. Recent work has identified the Toll-like receptors as important members of the PRR family. These receptors initiate defensive responses upon detection of pathogenic microorganisms. In most cases, these responses lead to resolution of infection but in others, they lead to deleterious inflammatory responses such as acute lung injury or possibly the inflammation of chronic diseases. The goal of this research is to understand the molecular basis for receptor recognition of PAMPs. Specifically, we propose two hypotheses: 1) that PAMPs interact directly with their cognate PRRs, and 2) that PRRs have more than one PAMP recognition site. To study these hypotheses we have formulated four specific aims: SAI: To synthesize specific cross linkable PAMPs for the relevant innate immune system PRRs. SA 2: To utilize cross linkable ligands to test for direct interactions between the PAMPs and the PRRs and to determine the specific sites of attachment of the PRRs to the PAMPs by mass spectrometric definition of the cross linking sites. SA 3: To test for the existence of multiple PAMP binding sites on a specific PRR by determining whether binding (or cross linking) of a given PAMP to its proposed PRR is competitive with or independent of a panel of other PAMPs specific for that PRR. SA 4: To devise mutational strategies to test a) whether sites of cross linking determined in Specific Aim 2 are functionally important in ligand binding and cellular activation and b) whether there are multiple binding sites for different PAMPs on single PRRs. By understanding how PRRs recognize PAMPs we hope to be able to propose methods for therapeutic manipulation of PAMP - PRR interactions.